The physical arrangement of genes and regulatory sequences in the genome has long been known to play a critical role in determining when and where a gene is expressed during development. Mutations that alter this arrangement often led to misregulation of genes which can have a profound impact on the survival of an organism. In development, Hox and globin gene clusters have served as the primary model for elucidating regulatory mechanisms responsible for coordinating the activity of linked genes. However, it is not known how widely these findings can be applied. Interestingly, in our own studies, we recently uncovered Mb-scale clustering of Gli1binding regions (GBRs) in the genome using chromatin immunoprecipitation combined with ultra-high throughput sequencing (ChlPSeq). These GBR dense regions correspond to two paralogous gene clusters on mouse chromosomes 2 and 12 that contain members of the Nkx, Pax and Fox gene families of transcription factors. Several of these factors: Nkx2-2, Nkx2-9, Nkx2-1, Pax1, Pax9, and Foxa2 are all known to be responsive to Sonic hedgehog (Shh) signaling during development yet act in different biological contexts. To investgate the significance of this clustering, I will use a series of integrated approaches to elucidate how target gene specificity is achieved within this set of linked genes. In situ hybridization and transgenic reoprter assays will be performed to determine the relationship between genomic sequence features and developmental expression patterns. I will assess GH1 and Gli3 binding profiles in sclerotomal cells using ChlPSeq. This analysis will be highly complementary to our neural tube and limb bud studies, and will provide additional Gli binding patterns across the regions. Lastly, I will investigate the 3D organization of this defined chromosomal region using the chromosomal conformation capture assay. This structural data will be critical for understanding how this set of linked genes is differentially regulated. The proposed studies have the opportunity to address the evolution of genomic regulatory architecture that is distinct from what has been done for single gene families. The elucidation of the transcriptional regulatory network controlled by Shh signaling in different contexts (e.gneural tube, limb bud and somites) will provide essential information for understanding how this important signaling pathway functions in development and disease as well as provide insight into principles of genome organization.